3,4-Methylenedioxymethamphetamine (MDMA, «ecstasy») 3' is a 'psychedelic amphetamine' that has gained popularity over the past 20 years because of its ability to produce strong feelings of comfort, empathy, and connection to others. MDMA analysis in blood and urine samples have been consistently used for clinical pharmacology studies and forensic science cases. However, new developments in clinical toxicology require new analytical approaches and the use of alternative biological matrices for establishing whether individuals have consumed the drug, when and/or if they have been acting under the effect of the drug. It is postulated that MDMA physic-chemical properties: (i) pKa of around 9. 9 corresponding to a weak base that facilitates the transfer of MDMA from plasma (pH=7. 4) to fluids/matrices with a favourable pH gradient, (ii) high liposolubility with volumes of distribution between 6 and 7 liters per kilogram, (iii) low protein binding, favour its distribution to biological matrices in humans. Several non-conventional biological matrices such as hair, sweat and saliva, because of drug accumulation due to its physico-chemical properties, might be of use for the detection of past and recent exposure to MDMA. Three different studies were set-up. The study 1 investigating the pharmacokinetics of MDMA in saliva after a single oral dose administration of 100 mg to eight healthy volunteers, the second investigating the pharmacokinetics of MDMA in sweat after a single dose administration of 100 mg to eight healthy volunteers, and finally a study on segmental analysis of MDMA in hair of thirteen drug consumers with different patterns of consumption. . The first study evidenced that MDMA is excreted in saliva, after a single 100 mg dose administration, with concentrations (range 1728. 9-6510 µg/ml at 1. 5 h after drug intake) one order of magnitude higher than those observed in plasma (range 134. 9-223 µg/ml at 1. 5 h after drug intake) and following a time course kinetics which parallels that of plasma and that of subjective effects and psychomotor performance. On-site testing by Drugwipe device proved suitable to detect individuals under the influence of drug effects in the first 6 hours after drug intake by non-invasive and rapid collection of salivary specimens. The second study showed that MDMA appears in sweat and can be quantified already in the first few hours after a single dose administration, when subjective effects are apparent (concentration range 3. 2-1326 ng/pacth). This result makes: the sweat patch technology useful for monitoring MDMA accumulation in sweat at least during the 24 hours after a single administration, On-site sweat testing by drugwipe device suitable to detect individuals under the influence of drug effects by non-invasive and rapid collection of minute amounts of sweat. MDMA appears in hair from consumers (concentration range 1. 2-12. 6 ng/mg hair) and can be detected in hair segments corresponding to the last one, six and twelve month of repeated drug use. For this reason: Hair analysis of MDMA can be used to evaluate exposure or abstinence to the drug in the last months, hair concentration of MDMA in different hair segments can predict levels of drug use(r2=0. 92) and can be eventually associated to chronic psychophysical effects induced by the repeated drug use. The measurement of MDMA in saliva is a valuable alternative to determination of plasma drug concentrations both in clinical and toxicological studies A common characteristic of the three different matrices is that the parent drug MDMA was always the principal, most abundant analyte detected, whose concentration could be associated with drug-induced effects and drug history. As already assessed, drug analysis in hair extends the information of drug consumption to a wider time-window than that of other non-invasive biological matrices, such as saliva and sweat. These latter two matrices can account for acute pharmacological effects induced by the drug, while results from hair testing can be used to assess repeated exposure to drug and eventual association with long term drug induced effects, such as neurotoxicity and psychological performance in the specific case of MDMA.